Summary: Conjugate vaccines of polysaccharides (PS; including lipopolysaccharides or LPS) utilize a carrier protein as a vehicle to convert the T cell-independent PS to a T cell-dependent protein-PS conjugate, and thus afford protection to an otherwise PS-non-responding population such as infants and the elderly. In this research, we investigate the issues related to the chemistries of conjugate vaccine synthesis and their impacts on vaccine immunogenicity and effectiveness. This includes the studies of: 1) the structures of PS and LPS, carrier proteins and peptides, spacer molecules and the conjugates, 2) conditions of various chemical reactions for conjugate formation, and 3) characterization of the conjugates in the aspects of physical and chemical properties and immunology. Group C meningococcal polysaccharide-tetanus toxoid conjugate (MC-TT) has been encapsulated in the microspheres of biodegradable polymers of poly lactic acid and poly lactic-co-glycolic acid by solid in oil in solid and solid in oil in water methods. It was found that the encapsulated MC-TT was progressively released in the in vitro release study. One injection immunization of the combined encapsulated and free MC-TT induced comparable anti-MC antibody as the traditional three-injection regimen at the dose of 2-3 ug per mouse. At the dose of 0.2-0.3 ug per mouse, the one-injection regimen elicited 3-4 times higher antibody response than the 3-injection administration. The induced antibody is biologically functional as shown by its high bactericidal titer. The encapsulated plus free MC-TT could be used to improve the efficiency of preventing diseased caused by group C N. meningitides. Determination of free polysaccharide content in conjugate vaccines is critical as the effective ingredient in conjugate vaccine is the conjugated polysaccharide. We have established the procedures in measuring the free polysaccharide the conjugate products of group C meningococcal and pneumococcal polysaccharides by the combination of micro-ultra-centrifugation, reverse phase C18 adsorption, HPLC and chemical analysis. We have synthesized PS-peptides conjugates for pneumococcal types 1, 3, 4, 5, 6B, 7F 9V, 14, 18C, 19F and 23F, and for meningococcal serogroups A, C, Y, and W135 using 11 T cell epitope peptides from published literatures. The immunogenicity of these singular and combined 4-valent (meningococcal), and 7-and 11-valent (pneumococcal) conjugates were investigated in mice in parallel to the corresponding pneumococcal PS-TT conjugates. It was found that in both singular and combined vaccine immunization, two of the 11 peptides are immunologically effective as carriers for all the 15 PS, and another two peptides were effective for more than half of the 15 PS. The elicited antisera will be tested for their opsonophagocytosis activity to the homologous pneumococci or bactericidal activity to the homologous meningococci. These PS-peptide conjugates may be useful for studying the effects of carrier suppression of conjugate vaccines, and interference in the combined vaccines. In other research, we have conjugated LPS from mutants of group B menigococci and B. pertussis to TT. These conjugates exhibit T-dependent immunogenicity against homologous LPS, and have a marked reduction in LAL activity. The bactericidal activities of the antibodies induced by these conjugates are currently being measured in vitro and in vivo. We have adapted and developed purpald assay methods for quantification of LPS and glycol-containing PS. These methods provide: 1) a convenient means (96-well tissue culture plates/room temperature vs. temperature higher than 90 degree/test tubes) for measurement of LPS and PS, 2) a measurement for LPS from Haemophilus influenzae, Bordetella pertussis, and Vibrio cholerae, 3) a determination of epitope preservation in conjugate vaccines.